Breast cancer patients die from metastatic disease. Primary breast adenocarcinoma spreads to distant organs by shedding circulating tumor cells (CTCs) in the blood. When these cells leave the systemic circulation, they convert into Disseminated Tumor Cells (DTCs), which are the seeds of secondary tumors. Mounting evidence indicates that existing metastases can mobilize cancer cells back into the blood, thus leading to further spreading that precipitates clinical progression. Thus, strategies aimed to impair tumor seeding have the potential to arrest or significantly decelerating the extension of the disease. Chemokine receptors have been implicated in dissemination, proliferation and survival of cancer cells. Our recent work indicates that the chemokine receptor CX3CR1 is over-expressed in both primary breast tumors and metastatic lesions and plays a role in the lodging of breast cancer cells to the skeleton of animal models. Furthermore, studies form others have shown that this receptor is expressed by cells with tumor-forming abilities and can transactivate growth factor receptors regulating proliferation and survival. Thus, based on the existing literature and our preliminary data, we hypothesize that CX3CR1 is implicated in both early and late stages of metastasis and that its blockade can both deter breast cancer cells from seeding multiple organs and counter their tumor- initiating properties. To test this hypothesis, we will use a combination of CRISPR interference (CRISPRi) and a novel small-molecule antagonist to delineate the time-frame for CX3CR1 involvement in metastatic progression and the effects of pharmacologic targeting of CX3CR1 in tertiary spreading from existing metastases. Finally, we intend to gain a mechanistic understanding of CX3CR1 signaling and determine the molecular pathways associated with tumor-initiation properties activated by this receptor in breast cancer cells. This proposal is structured into three specific aims: AIM 1. To delineate the temporal involvement of CX3CR1 in metastatic progression; AIM 2. To define the effects of CX3CR1 blockade on tumor re- seeding from existing metastases; AIM 3. To elucidate the mechanistic details of CX3CR1 involvement in the metastatic behavior of breast cancer cells. This proposal will define how CX3CR1 influences breast cancer metastatic behavior, reveal important mechanistic details of its activity in cancer cells and provide pre-clinical support for the pharmacologic targeting of this receptor, which presents a high therapeutic potential from several standpoints. From a drug-safety perspective, transgenic mice knockout for CX3CR1 are viable and exhibit no impairment of their immune response under unchallenged conditions, overt behavioral abnormalities or macroscopic anatomical alterations. Thus, if successful our work should pave the way to a novel series of therapeutics and promote multifaceted strategies to counteract the metastatic progression of breast cancer.